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Abstract

Background

The sirtuins are a conserved family of NAD+-dependent histone/protein deacetylases that regulate numerous cellular processes,
including heterochromatin formation and transcription. Multiple sirtuins are encoded
by each eukaryotic genome, raising the possibility of cooperativity or functional
overlap. The scope and variety of chromatin binding sites of the sirtuins in any specific
organism remain unclear.

Results

Here we utilize the ChIP-seq technique to identify and functionally characterize the
genome-wide targets of the sirtuins, Sir2, Hst1 to Hst4, and the DNA binding partner
of Hst1, Sum 1, in Saccharomyces cerevisiae. Unexpectedly, Sir2, Hst1 and Sum1, but not the other sirtuins, exhibit co-enrichment
at several classes of chromatin targets. These include telomeric repeat clusters,
tRNA genes, and surprisingly, the open reading frames (ORFs) of multiple highly expressed
RNA polymerase II-transcribed genes that function in processes such as fermentation,
glycolysis, and translation. Repression of these target genes during the diauxic shift
is specifically dependent on Sir2/Hst1/Sum1 binding to the ORF and sufficiently high
intracellular NAD+ concentrations. Sir2 recruitment to the ORFs is independent of the canonical SIR complex
and surprisingly requires Sum1. The shared Sir2/Hst1/Sum1 targets also significantly
overlap with condensin and cohesin binding sites, where Sir2, Hst1, and Sum1 were
found to be important for condensin and cohesin deposition, suggesting a possible
mechanistic link between metabolism and chromatin architecture during the diauxic
shift.

Conclusions

This study demonstrates the existence of overlap in sirtuin function, and advances
our understanding of conserved sirtuin-regulated functions, including the regulation
of glycolytic gene expression and condensin loading.

Background

The sirtuins are a highly conserved family of NAD+-dependent protein deacetylases that regulate a wide range of cellular processes impacted
during aging and in age-associated diseases such as type 2 diabetes and cancer (reviewed
in [1]). They utilize NAD+ as a co-substrate during the deacetylation reaction, such that one molecule of NAD+ is converted into nicotinamide and 2'O-acetyl ADP-ribose for every lysine that is
deacetylated [2,3]. As a result, sufficiently high NAD+ concentrations are required to properly regulate cellular processes in which the sirtuins
participate [4,5]. This link to NAD+ gives sirtuins an inherent ability to 'sense' the intracellular energy status, and
regulate target proteins via lysine deacetylation.

The genomes of eukaryotic organisms usually encode multiple sirtuin proteins. For
example, there are seven sirtuins in mammals known as SIRT1 through SIRT7 [6], while the budding yeast Saccharomyces cerevisiae encodes five, known as Sir2, and Hst1 through Hst4 (homologs of Sir two) [7]. Sir2 is the founding family member [7], and was initially characterized as a factor required for transcriptional silencing
at the HML and HMR silent mating-type loci, telomeres, and the ribosomal DNA (rDNA) tandem array, each
of which have characteristics of heterochromatin in more complex eukaryotes (reviewed
in [8]). Eventually, Sir2 was found to be a histone deacetylase [9,10], a seminal discovery that instantly provided a mechanistic role for Sir2 in the formation
of heterochromatin. Equally exciting were the implications for aging because Sir2
was also characterized as a limiting factor for replicative life span [11], which is defined as the number of times a yeast mother cell divides before senescing.
Deletion of SIR2 shortens replicative life span, while increased SIR2 gene dosage extends both mean and maximum replicative life span [11]. Similarly, SIRT6 knock out mice prematurely age [12], and male SIRT6 transgenics are long-lived [13], suggesting that longevity and/or health span support by sirtuins could be one of
their conserved features.

Many histone-modifying enzymes are catalytic subunits of large multi-protein complexes,
and the nuclear sirtuins appear to follow this trend. At the HM loci and telomeres, yeast Sir2 is associated with the Sir3 and Sir4 proteins in a
complex known as SIR [14-16]. Deleting either of the SIR complex subunits results in a loss of transcriptional
silencing [17,18]. At the rDNA locus, Sir2 associates with Net1 and Cdc14 in the nucleolar silencing
complex known as RENT, which silences RNA polymerase II transcription from the intergenic
spacers [19,20]. The Sir2 paralog, Hst1, forms a complex with Sum1 and Rfm1 (the Sum1 complex), which
represses specific genes through localized histone deacetylation at promoters [21-23]. SIRT1 is considered the mammalian Sir2 ortholog, and like Sir2, is a histone deacetylase
that can function in heterochromatin formation [24]. SIRT1 also regulates the expression of numerous genes and deacetylates numerous
non-histone protein targets, but it has only recently been considered part of a larger
multi-protein co-repressor complex [25]. Thus, sirtuins are generally recruited to multiple targets (chromatin or non-histone)
through interactions with partner proteins that provide an added level of binding
specificity. Given such diversity, complete inventories of conserved sirtuin targets,
both chromatin and non-chromatin, are needed in order to understand how these factors
are truly integrated with various cellular processes via NAD+.

In this study we have focused on the identification and characterization of novel
chromatin targets of sirtuins in yeast, with the goal of uncovering additional cellular
pathways that are impacted by alterations in cellular NAD+, and determining the extent of functional overlap between the various sirtuins. Conserved
targets of this nature are also more likely to be mediators of longevity. We have
utilized chromatin immunoprecipitation (ChIP), followed by next generation DNA sequencing
(ChIP-seq), to obtain high-resolution chromatin association maps for each yeast sirtuin.
Work presented in this study functionally characterizes the overlapping roles for
Sir2, Hst1, and Sum1 in telomere maintenance, as well as NAD+-dependent repression of specific genes that are downregulated during the diauxic
shift, a time in which yeast shift their metabolism from aerobic fermentation to respiration.
These include genes involved in glycolysis, fermentation, and translation. Sir2, Hst1,
and Sum1 were also required for the efficient recruitment of condensin and cohesin
onto multiple shared target sites, including tRNAs and other genes downregulated during
the diauxic shift.

Results

From an earlier expression profiling study, we identified thiamine biosynthesis genes
as being upregulated when yeast cells were treated with the sirtuin inhibitor nicotinamide,
or when intracellular NAD+ levels were reduced by deleting the NAD+ salvage pathway gene NPT1 [26]. Hundreds of other genes were also upregulated, including known Hst1/Sum1 targets
such as middle sporulation and NAD+ biosynthesis genes. These results raised the question of whether each sirtuin specifically
regulates a distinct set of gene targets? We therefore set out to obtain a genome-wide
picture of binding sites for each sirtuin using ChIP-seq. The sirtuins were carboxy-terminally
tagged either with 13 copies of the Myc epitope (Sir2, Hst1, and Hst2) or one copy
of the TAP-tag (Hst3 and Hst4). Sum1 was also tagged with 13xMyc to test if it consistently
tracked with its binding partner Hst1. Cells growing exponentially in rich YPD medium
were then subjected to ChIP with anti-Myc or anti-TAP antibodies, and the recovered
DNA sequenced. Certain genomic regions were previously shown to be over- or under-represented
when genomic DNA was sequenced by this method [27]. Therefore, to control for representation in the ChIP libraries, crosslinked DNA
that went into the immunoprecipitation (IP) reactions (the input) was also sequenced.

Sirtuin binding at the silent mating-type loci and the rDNA tandem array

We first analyzed Sir2 at the silent mating-type loci HML and HMR to confirm the ChIP-seq procedure was effective. Sir2 was clearly enriched at the
HML-E and -I silencers as expected (Figure 1a). Sequence reads derived from repetitive regions are discarded by the mapping program,
so there is a lack of binding information for the α1 and α2 genes within HML because they are duplicated at MATα. However, Sir2 was still noticeably enriched between the two silencers (Figure 1a), which supported the results of an earlier study suggesting unidirectional spreading
from each silencer [28]. At HMR we observed a strong peak of Sir2 at the HMR-E silencer that extended rightward across the body of HMR as expected, but there was no corresponding peak at the HMR-I silencer (Figure 1b). Instead, a very strong peak of Sir2 was located at an adjacent tRNAThr gene, which had already been well characterized as a silencing boundary element [29]. Earlier ChIP results showed that Sir2 does not associate with HMR-I when the E silencer is defective, suggesting that Sir2 is not independently recruited to the
I silencer, but rather spreads all the way across HMR until it reaches the boundary [30]. Our data suggest an extension of this model in which the tRNAThr gene plays a dual role as a boundary element and as a protosilencer where Sir2 is
independently recruited. A protosilencer is defined as a cis-acting sequence that cannot establish silencing on its own, but instead cooperates
in establishment and maintenance of the silent chromatin [31]. Support for such a protosilencer model comes from a recent study where the tRNAThr boundary element was found to impose the cell cycle progression requirement for establishing
silencing at HMR [32].

Figure 1.High resolution ChIP-seq mapping at known Sir2, Hst1, and Sum1 targets. (a) Sir2 enrichment at the HML. Locations of the E and I silencers are indicated. (b) Sir2 enrichment at the HMR. The right boundary element tRNAThr gene is indicated as tT(AGU)C. Locations of the E and I silencers are indicated. (c) Sir2, Hst1, and Sum1 enrichment at the BNA2 and SPS4 promoters. (d) Sir2 enrichment at the rDNA repeats on chromosome XII. The left-most and right-most
repeats annotated in the SGD genome assembly are displayed, but the read counts in
brackets are a compilation from all approximately 175 repeats. The 5S genes within
the intergenic spacers are indicated as RDN5-1 and RDN5-2, and define the right end of each repeat.

There was no visible enrichment of Hst2, Hst3 or Hst4 at the silencers or across HML or HMR, but surprisingly, Hst1 and Sum1 were both highly enriched and co-localized with
Sir2 at the tRNAThr boundary element flanking HMR (Figure S1 in Additional file 1), suggesting they could also potentially be involved in the cell cycle progression
requirement in silencing linked to this cis-acting element. Given the unexpected co-localization of Sir2, Hst1, and Sum1 at the
tRNAThr gene, we next asked whether Sum1 and Hst1 were properly associating with several of
their known targets, including the promoters of specific NAD+ biosynthesis and middle sporulation genes [21,33]. As shown in Figure 1c, Sum1 was clearly bound to such promoters (BNA2 and SPS4) with a single strong peak and without any indication of spreading, consistent with
previous low-resolution ChIP assays [34]. Hst1 was also highly enriched at the same sites, but its occupancy was extended
a short distance (one or two nucleosomes) in both directions (Figure 1c). Importantly, Sir2 does not repress these genes [35], and we did not detect any binding signal in the ChIP-seq profiles (Figure 1c). Taken together, these results indicated that the ChIP-seq data were highly specific,
and the co-association of Sir2, Hst1, and Sum1 at the tRNAThr gene and other genomic sites (see below) is likely meaningful.

Sir2-dependent transcriptional silencing also occurs at the rDNA tandem array [36,37], which consists of approximately 150 to 200 identical rDNA gene copies on the right
arm of chromosome XII. Only the leftmost and rightmost repeats are annotated, so ChIP-seq
reads that map to these two repeats are a compiled average for the entire array (Figure
1d). The mapping program parameters were adjusted in this case to allow for repeated
sequences, so the read counts for Sir2 peaks in the rDNA approached 200,000, compared
to approximately 2,000 for HML and HMR. Consistent with previous silencing and ChIP results [38,39], Sir2 strongly associated with the rDNA intergenic spacers (IGS1 and IGS2), and its
extended enrichment to the left of IGS2 occurs in the same direction as Pol I transcription
(Figure 1d). The strongest Sir2 peak in IGS1 corresponded exactly to a replication fork block
site (chromosome XII coordinates: 460530-460570), where Fob1 recruits the RENT and
cohibin complexes [40]. While there may be some low-level association of Hst1, Sum1, Hst2, Hst3, and Hst4
across the rDNA, it was minimal compared to the overwhelming Sir2 signal (Figure S1
in Additional file), which is presumably derived from the RENT complex.

Telomere length maintenance by Sir2, Hst1, and Sum1

The third known type of silencing in yeast occurs at telomeres. The SIR complex is
recruited to telomeres through physical interaction with the Rap1 protein, which directly
binds to telomeric TG1-3 repeats [41,42]. As with the HM loci, sequence read coverage of many telomeric regions was sparse. When there was
sufficient coverage, Sir2 association with the terminal TG1-3 repeats was observed (Figure 2a), but its relative distribution among different telomeres was quite variable (Table
S1 in Additional file 1). Surprisingly, strong peaks of Hst1 and Sum1 binding were observed directly overlapping
with Sir2 at the TG1-3 repeats, regardless of whether they were terminal or internal clusters (Figure 2a,b; Table S1 in Additional file 1). From binding sites identified using BayesPeak [43], we calculated the enrichment of Hst1, Sum1, or Sir2 for telomeric repeats was at
least 30-fold greater than expected by chance (Table S2 in Additional file 1). Sir2 enrichment at terminal and internal TG1-3 clusters typically extended inward up to approximately 1 kb, whereas Hst1 and Sum1
were more restricted to the actual repeats (Figure 2a,b).

Figure 2.Functional sirtuin enrichment at telomeric repeat clusters. (a) Enrichment of Sir2, Hst1 and Sum1 at terminal TG1-3 repeats at the left telomere of chromosome XV (Tel_ChrXV_L). The black rectangle represents
the end of the chromosome and the circle represents the centromere. (b) Enrichment of Sir2, Hst1 and Sum1 at an internal TG1-3 subtelomeric cluster on the right arm of chromosome VIII (Tel_ChrVIII_R). (c) Quantitative ChIP assays measuring the effect of a rap1-17 mutation on telomeric Sir2-myc enrichment (left panel) or Hst1-myc and Sum1-myc enrichment
(right panel). The amplified region was either 70 bp or 7,000 bp away from the terminal
TG1-3 cluster on the right arm of chromosome VI. The immunoprecipitated PCR signal is relative
to the input chromatin PCR signal (Relative ChIP). In this experiment the untagged
background signal was subtracted out. Error bars represent standard deviation. (d) Southern blot detection of telomere (TEL) lengths using a probe that hybridizes to
poly (TG1-3) sequences. WT, wild type.

To test whether Hst1 and Sum1 were recruited to telomeric repeats via Rap1, their
enrichment was assayed by quantitative ChIP in wild type (WT) and rap1-17 mutant strains [44]. The rap1-17 allele truncates the carboxy-terminal SIR interaction domain of Rap1, resulting in
mis-localization of the SIR complex and severe silencing defects at the HM loci and telomeres [42,45]. The repetitive nature of telomeric TG1-3 sequences prevented direct real-time PCR amplification of these regions, so we instead
assayed 70 bp adjacent to the cluster on the right arm of chromosome VI, or 7,000
bp away from the repeats as a control. As expected, strong telomeric enrichment of
Sir2 at the 70 bp position was eliminated in the rap-17 mutant (Figure 2c, left panel). There was also a low level of Hst1 and Sum1 enrichment 70 bp from the
TG repeats in the WT strain, confirming the poor spreading capability of these two
proteins, relative to Sir2. Importantly, even this low level Hst1 and Sum1 occupancy
was significantly reduced in the rap1-17 mutant (Figure 2c, right panel), suggesting that Rap1 significantly contributes to their recruitment.
However, we cannot rule out the possibility that occupancy was indirectly reduced
in the mutant because of the general defect in telomeric heterochromatin formation.

Given the precise association of Sir2, Hst1, and Sum1 with terminal TG repeats, we
next tested for functional consequences of deleting the sirtuins or SUM1 on telomere length. As shown in Figure 2d, sir2Δ or hst1Δ mutants had little effect on telomere lengths compared to WT strains, but the sir2Δ hst1Δ double mutant clearly had shorter telomeres on average, indicating significant redundancy
between the two paralogs. The sum1Δ mutant also had short telomeres similar to the double mutant length. Compared to Sir2,
Hst1, and Sum1, the occupancy of Hst2, Hst3, and Hst4 at the TG1-3 repeats was absent or extremely low (Figures 2a,b), suggesting the slightly longer telomeres in hst2Δ and hst3Δ mutants were likely an indirect effect (Figure 2d). Because Sir2, Hst1, and Sum1 appeared to have more specific co-enriched binding
sites and functional redundancy than initially anticipated, we focused subsequent
analysis on these three proteins.

Sir2, Hst1, and Sum1 associate with and regulate highly transcribed genes that are
downregulated during the diauxic shift

Comparisons of BayesPeak-identified Sir2, Hst1 or Sum1 enrichment peaks to gene annotations
revealed that the largest percentage of binding sites for each protein was surprisingly
found within ORFs (Figure 3a). To determine if there was any relationship between ORF enrichment and expression
levels for the associated genes, composite plots were generated for Sir2, Hst1, Sum1,
or Hst2 ChIP-Seq read numbers across all yeast genes, with the expression levels divided
into five quintiles (Figure 3b). Sir2, Hst1, and Sum1 enrichment, but not Hst2, was strongest toward the 3' end
of ORFs with the highest expression levels (5th quintile). There was also significant
overlap between the ORFs bound by each factor (Figure 3c), which was easily visualized in the genome browser for highly expressed genes such
as PDC1, ENO2, and CDC19 (Figure 3d). Traditional ChIP assays were performed with several target genes to confirm the
ORF enrichment was not a sequencing artifact (Figure 3e; Figure S2a in Additional file 1). Hst2 did not have this pattern of binding in the composite plots or in the genome
browser, and was not ORF-enriched in standard ChIP assays compared to an untagged
control (Figure 3e), consistent with its reported cytoplasmic localization pattern [46].

Figure 3.Summary of Sir2, Hst1, and Sum1 binding across the yeast genome. (a) Gene-based distribution of Sir2, Hst1, and Sum1 binding sites identified with BayesPeak.
Filled circles represent relatively small ORFs that were completely embedded within
a peak. (b) Composite plots showing that Sir2, Hst1, and Sum1, but not Hst2, are enriched at the
most highly expressed genes. The sequence of each annotated ORF in the genome was
normalized to 1,000 bins, with another 500 bins upstream and 500 downstream, into
which the number of sequencing reads (tags) were distributed. The expression level
of each ORF was divided into five quintiles, with the 5th quintile the highest. (c) Venn diagram showing the overlap between genes with Sir2, Hst1 or Sum1 are enriched
across >60% of the ORF. **P-value <0.005. (d) Screenshots of Sir2, Hst1, Sum1, and Hst2 enrichment across the PDC1, ENO2, and CDC19 genes. Hst1 acts as a negative control similar to the Input sequence. (e) ChIP assay confirming the enrichment of Sir2, Hst1 and Sum1 across the ORFs of PDC1 and ENO2. Relative IP indicates the ratio of IP PCR signal to the input chromatin PCR signal
for each sample. Error bars represent standard deviation.

Many of the most highly expressed genes during log phase tend to be strongly repressed
when cells enter the diauxic shift. We used publicly available expression profiling
data [47], to ask whether genes with Sir2, Hst1, or Sum1 binding on their ORFs were differentially
regulated during the diauxic shift (Figure S2b in Additional file 1). For each individual factor there was a trend for association with downregulated
genes, which reached strong statistical significance for Hst1. Coupled with this trend,
there was also a highly significant depletion for upregulated genes (Figure S2b in
Additional file 1,). Furthermore, genes with overlapping Sir2, Hst1, and Sum1 ORF binding were enriched
for Gene Ontology terms related to glycolysis, glucose fermentation, translation,
and cell wall biosynthesis (Table S3 in Additional file 1), all processes that are highly active during log phase and then repressed during
the diauxic shift [48]. Five of the ORF-targeted genes encode enzymes involved in glucose fermentation (Figure
4a, highlighted in red), so we hypothesized that expression of such genes would be dysregulated
when SIR2, HST1, or SUM1 were deleted. No significant differences in PDC1 or ENO2 expression were observed in the deletion mutants during log phase (Figure 4b; Figure S3a in Additional file 1), but they were not properly repressed when entering the diauxic shift (Figure 4c; Figure S3b in Additional file 1). PDC1 and ENO2 in the WT strain were strongly repressed within 6 hours after log phase, while repression
in the mutants was significantly delayed. This was especially true in the sir2Δ hst1Δ double mutant, which was unable to fully repress PDC1 even 10 hours after log phase (Figure 4d), suggesting some redundancy between Sir2 and Hst1, as was also observed in telomere
length regulation (Figure 2d).

Figure 4.Repression of PDC1 by Sir2 and Hst1 during the diauxic shift. (a) Overview of the glycolysis/fermentation pathway in yeast. Red lettering indicates
genes bound by Sir2, Hst1 and Sum1 across the ORF. Bold lettering indicates the genes
only bound by Sir2. (b) Quantitative RT-PCR analysis of PDC1 mRNA levels in log phase WT and mutant strains. Expression level in the WT strain
was normalized to 1.0. (c) PDC1 expression level in WT and deletion strains when progressing through the diauxic shift.
(d) Enlarged image of (c) showing the later time points. Error bars represent standard
deviation.

During the diauxic shift, cellular metabolism switches from fermentation to respiration,
which causes an increase in the NAD+/NADH ratio. We observed this increased ratio in the WT strain within 3 hours post
log phase (Figure 5a). Since the higher NAD+/NADH ratio induced by calorie restriction has been reported to promote Sir2 function
[49], we next tested whether simply reducing the intracellular NAD+ concentration by deleting NPT1 would also delay PDC1 repression, which it did (Figure 5b). Similar results were observed for ENO2 and CDC19 (Figure S3c in Additional file 1). The time course of glucose consumption from the YPD medium was almost identical
between the WT strain and the sir2Δ, hst1Δ, or sum1Δ mutants (Figure 5c), but it was still formally possible that the mutants were not properly repressing
PDC1 simply because their entry into the diauxic shift was delayed. If this were the case,
then other genes down-regulated at the diauxic shift whose ORFs were not bound by
Sir2, Hst1, or Sum1 should also show a delay in repression in the mutant strains.
PDC5 encodes a minor isoform of pyruvate decarboxylase (Figure 4a), but does not have Sir2, Hst1, or Sum1 enriched on the ORF (Figure 5d). Importantly, PDC5 repression at the diauxic shift occurred normally in the sir2Δ, hst1Δ, or npt1Δ mutants (Figure 5e). To make sure this was not a peculiarity of PDC5, we tested three additional genes from this class (CLB1, RIF1, and RPB3) in the sir2Δ and npt1Δ mutants, and again did not observe any defect in repression across the time course
(Figure S4 in Additional file 1). These results suggest a model where Sir2 and Hst1 are associated with specific
targeted ORFs in a poised state during log phase, and then become functional for transcriptional
repression at onset of the diauxic shift, perhaps aided by the increased NAD+/NADH ratio. This would result in the observed early and rapid gene repression, probably
before the overall reduction in the general transcription machinery occurs.

We initially hypothesized that Sir2 and Hst1 function in repression of their ORF-targeted
genes by deacetylating histones across the ORF when cells enter the diauxic shift.
However, using quantitative ChIP, there was no consistent increase in various histone
H3 or H4 acetylation marks on the PDC1 or ENO2 genes in sir2Δ, hst1Δ, sum1Δ, or npt1Δ mutants during log phase or the diauxic shift (data not shown). Thinking that there
could be some redundancy involved, sir2Δ sum1Δ and sir2Δ hst1Δ double mutants were also tested, but H3 or H4 acetylation was still not elevated compared
to WT (Figure S5 in Additional file 1). Even so, the requirement for high NAD+ concentration in Figure 5b strongly suggested that Sir2 and/or Hst1 catalytic activity was required for the
repression. The effect of an H364Y mutation in SIR2 that eliminates deacetylase activity was tested and found to block PDC1 repression at onset of the diauxic shift (Figure 5f), similar to the sir2 deletion. Therefore, the mechanism of repression remains unknown, but current evidence
points toward either non-traditional lysine modifications or non-histone deacetylation
targets such as RNA polymerase or elongation factors being important (see Discussion).

Evidence for novel Sir2 and Hst1 complexes on targeted ORFs

The apparent inactivity of Sir2 and Hst1 on histones at the targeted ORFs suggested
they could be functioning independently of their canonical HDAC complexes at these
sites. To check this possibility, we first tested whether Sir2 or Hst1 enrichment
on the PDC1 ORF was altered in a sum1Δ mutant. Sir2 enrichment was surprisingly lost, while Hst1 was increased (Figure 6a), strongly suggesting that Sum1 is involved in recruiting Sir2, but not Hst1. We
next tested whether Sir2 or Hst1 enrichment was altered in sir3Δ or sir4Δ mutants. As shown in Figure 6b, Sir2 enrichment was maintained in both mutants, and actually increased in the absence
of SIR4, reminiscent of the increased nucleolar Sir2 localization and rDNA silencing in a
sir4Δ mutant caused by redistribution of the telomeric Sir2 pool [50,51]. Similarly, increased Hst1 enrichment in the sum1Δ mutant could be due to release of Hst1 from its traditional promoter targets. Since
Sum1 appeared to be recruiting Sir2, we thought Hst1 may behave oppositely as well,
and be recruited via interactions with Sir4. However, Hst1 enrichment at PDC1 was unaffected in the sir4Δ mutant (Figure 6c). Taken together, these ChIP results suggest a model whereby Sir2 is recruited to
its ORF targets via Sum1, while Hst1 is recruited through an unknown bridging factor
(Figure 6d). It is important to note that independent mechanisms of Sir2 and Hst1 recruitment
are consistent with their observed functional redundancy in gene repression during
the diauxic shift (Figure 4).

Sir2, Hst1, and Sum1 promote condensin and cohesin recruitment to multiple chromatin
loci, including tRNA and other diauxic shift-repressed genes

We earlier noted significant enrichment of Sir2, Hst1, and Sum1 at the tRNAthr boundary element adjacent to HMR-I (Figure 1b). Subsequent binding site identification on Pol III-transcribed genes using BayesPeak
revealed fold enrichment values of between 110- and 169-fold over random expectation
(Table S4 in Additional file 1). There was also significant overlap between the Sir2, Hst1, and Sum1 associated
genes (Figure S6 in Additional file 1). Example Integrative Genomics Viewer (IGV) screenshots for tQ(UUG)H, tE(UUC)E1,
and SNR30 binding are shown in Figure 7a, revealing that these small genes are often the center of a broader peak for all
three proteins. Binding to tQ(UUG)H and SNR30 was confirmed by standard ChIP assays (Figure 7b).

Figure 7.Co-localization of Sir2, Hst1, and Sum1 with cohesin and condensin at Pol III-transcribed
genes. (a) IGV screenshots showing enrichment of Sir2, Hst1, and Sum1 with three Pol III-transcribed
genes. Hst2 behaves as a negative control. (b) ChIP assay confirming the enrichment of Sir2, Hst1 and Sum1. (c) The decreased binding of a cohesin subunit (Mcd1) and two condensin subunits (Smc4
and Brn1) to the tRNA genes when SIR2, HST1 or SUM1 are deleted. Cells were grown in YPD to the log phase. The relative IP in each panel
indicates the IP PCR signal divided by the input chromatin PCR signal, for normalization.
Error bars represent standard deviation.

tRNA genes and the associated TFIIIC binding sites are well established as being chromatin
insulator and boundary elements in yeast and human cells [29,52]. In yeast, the condensin and cohesin complexes have previously been demonstrated
through ChIP-chip experiments to globally associate with tRNA genes [53,54]. We were curious if there was significant overlap of Sir2, Hst1, or Sum1 sites with
the published cohesin or condensin binding sites. Indeed, the peaks for all three
tagged proteins showed significant overlap with condensin peaks (approximately 140-
to 200-fold over random) and cohesin peaks (approximately 24- to 29-fold over random)
(Table S5 in Additional file 1). Sir2 and Hst1 were previously shown to function in cohesin recruitment to the silenced
rDNA and HMR loci in yeast [55,56], but such a relationship with condensin had not been reported. To test whether Sir2,
Hst1, and Sum1 are involved in the loading of condensin and/or cohesin onto tRNA genes,
the condensin subunits Brn1 and Smc4, and the cohesin subunit Mcd1 were carboxy-terminally
tagged with the 13xMyc epitope in WT, sir2Δ, hst1Δ, and sum1Δ strains. Steady state protein expression was equivalent in all the strains, as measured
by western blotting with the 9E10 α-Myc antibody (Figure S7a in Additional file 1). ChIP assays for the tagged subunits revealed a strong dependence for Sir2, Hst1,
and Sum1 in loading both condensin and cohesin onto tQ(UUG)H and tE(UUC)E1 (Figure
7c).

The links between Sir2, Hst1, and Sum1 with condensin and cohesin were not limited
to Pol III-transcribed genes. Pol II-transcribed genes such as PDC1 with Sir2, Hst1, and Sum1 coating the ORF often had a condensin peak located near
the 3' end of the gene (Figure 8a). This was intriguing given the trend for Sir2, Hst1, and Sum1 association toward
the 3' ends of genes (Figure 3b). Quantitative ChIP demonstrated that condensin (Brn1 and Smc4) and cohesin (Mcd1)
were indeed highly enriched in the intergenic region 3' of PDC1 and ENO2 (Figure 8b). But interestingly, significant levels of association were also observed across
the ORFs. Regardless of the location tested, or the level of enrichment, the sir2Δ, hst1Δ, and sum1Δ mutations impaired the condensin/cohesin recruitment (Figure 8b), which was also observed for targeted ribosomal protein genes (Figure S7b in Additional
file 1). We were unable to detect direct physical interactions between Sir2 and tagged condensin
subunits through co-IP assays (data not shown), suggesting the recruitment may not
be mediated by a direct physical interaction, but perhaps related to Sir2 catalytic
activity. Consistent with that idea, the npt1Δ mutant with reduced NAD+ caused a partial defect in cohesin (Mcd1) or condensin (Smc4) subunit association
at the rDNA (Figure 8c). Furthermore, loss of Npt1 reduced enrichment of a cohesin loading factor subunit
(Scc2), which may also promote functional condensin association with chromosomes [53]. Since condensin and cohesin mediate long-range chromatin interactions, this suggests
that sirtuins could potentially make upstream contributions to this type of chromatin
organization. Potential implications are addressed in the discussion.

Figure 8.Enrichment of cohesin and condensin at the PDC1 gene and rDNA is dependent on SIR2, HST1, SUM1 and NPT1. (a) Zoomed out view of a 100 kb chromosome XII region with four ORFs with Sir2, Hst1,
and Sum1 enrichment that coincide with a condensin peak (asterisks). (b) The reduced binding of cohesin and condensin subunits to the PDC1 ORF when SIR2, HST1 or SUM1 was deleted. Cells were grown in YPD to log phase. (c) Reduced association of cohesin (Mcd1) and condensin (Smc4) subunits to the rDNA when NPT1 was deleted. Scc2 is a subunit of the Scc2/4 cohesin loading complex. The relative
IP in each panel indicates the IP PCR signal divided by the input chromatin PCR signal,
for normalization. Error bars represent standard deviation.

Discussion

Overlapping functions of Sir2 and Hst1 mediated by Sum1

One of the unexpected findings from this study was the large number of locations where
Sir2, Hst1, and Sum1 were co-enriched. Sir2 and Hst1 are paralogs that have acquired
differential functions, but are still similar enough to substitute for one another
under specific circumstances [35]. For example, a dominant SUM1-1 mutation suppresses the HM silencing defect of strains deleted for SIR genes by directing Hst1 to the silencers [57,58]. HST1 overexpression can also suppress the HM silencing defects of a sir2Δ mutant [7], and Sir2 can partially substitute for Hst1 in an hst1Δ background to suppress middle sporulation genes during vegetative growth [35]. It should be noted that Sum1 has repressive activity at the HMR-E silencer and some meiosis genes that is independent of Hst1 [33,59]. Perhaps Sum1 interacts with Sir2 at such locations when Hst1 is missing, which could
even be mediated by another adaptor protein, similar to the role that Rfm1 plays in
facilitating Hst1 interactions with Sum1 [22]. Our unexpected finding that Sum1 is required for Sir2 enrichment at the PDC1 ORF supports this hypothesis, and reveals even more diversity in the mechanism of
chromatin targeting for Sir2 than was anticipated. However, the Sir2 recruited to
ORFs via Sum1 is likely a minor subset of the overall Sir2 population, as indicated
by the relatively lower Sir2-myc ChIP-seq read counts at ORFs compared to telomeres,
the rDNA, and HM loci. Furthermore, Sir2 interaction with Sum1 in co-IP experiments has only been observed
when HST1 is deleted [35], implying that most Sir2 is not associated with Sum1. Increased Sir2 enrichment in
the sir4Δ mutant is likely due to redistribution from telomeres, and suggests the targeted ORFs
are in competition with telomeres and rDNA for limiting Sir2. The mechanism of Sum1
and Hst1 recruitment to specific ORFs remains unknown, and merits future investigation.

We were initially surprised to detect strong Hst1 and Sum1 binding to the telomeric
TG1-3 repeats, but genome-wide Southern blotting screens for deletion mutants with altered
telomere lengths independently identified sum1Δ as having short telomeres [60,61]. Our results confirm the role of Sum1 in telomere maintenance, but also implicate
Sir2 and Hst1, which appear to compensate for each other when one or the other is
deleted. It had been speculated that the effect of the Sum1 complex on telomere length
was likely an indirect effect on expression of upstream regulators of telomere length
[61]. However, the precise association of Sum1 and Hst1 with the TG repeats strongly suggests
that the SIR and Sum1 complexes have more direct functions in protecting the telomere
ends. Rap1 recruits the SIR complex to telomere repeats [42], and we have now shown that Hst1 and Sum1 telomeric recruitment also depends on Rap1.
Interestingly, there is a positive genetic interaction (P = 1.7e-06) between RAP1-damp and sum1Δ alleles in the DRYGIN database [62], supporting the idea of a functional relationship.

tRNAs, sirtuins, and repression of adjacent Pol II-transcribed genes

We previously reported that Hst1 and Sir2 both directly repress basal expression of
the thiamine biosynthesis gene THI4 by binding to and deacetylating histone H4 at a region approximately 700 to 800 bp
upstream of the transcription start site [26]. That region happens to overlap with the tG(UCC)G gene, which, like many other Pol
III-transcribed genes in the dataset, is co-enriched for Sir2, Hst1, and Sum1 binding
(Table S4 in Additional file 1, and data not shown). We now hypothesize the tG(UCC)G gene is a cis-acting repressive element in this context, and that other tRNA genes with Sir2, Hst1,
and Sum1 co-binding may repress adjacent Pol II-transcribed genes under specific growth
conditions. Not all tRNA genes showed strong Sir2/Hst1/Sum1 co-enrichment, so any
repressive effect would be limited to a subset of tRNA genes, including those that
are within sufficient distance to a susceptible promoter, like THI4. Consistent with this model, an earlier genomic analysis found that Pol II-transcribed
genes with a tRNA gene located within approximately 400 bp of the promoter were expressed
approximately 3.5-fold less than genes without an adjacent tRNA gene [63]. A more recent study also showed that tRNA genes are often associated with Sir3 and
Sir4, and those with strong DNA replication pausing activity can enhance silencing
when positioned adjacent to an ADE2 reporter gene flanked by the HMR-E and -I silencers [64].

The Engelke lab previously described a form of tRNA gene-mediated (TGM) silencing
where the SUP4 or SUP53 tRNA genes repress expression of an adjacent HIS3 reporter gene [65]. This silencing is dependent on the clustering of tRNA genes at the nucleolar periphery,
but does not appear related to HM, telomeric, or rDNA silencing, because deleting the SIR genes has no effect [54,66]. Instead, TGM silencing is dependent on the association of condensin and cohesin
with tRNA genes [54,67]. It is therefore intriguing that deletion of SIR2, HST1, or SUM1 causes a reduction in condensin and cohesin association with targeted tRNA genes (Figure
7c), but sir2Δ does not affect TGM silencing when tested with the reporter gene system [66]. The enrichment of Sir2, Hst1, and Sum1 is relatively weak at SUP4 and SUP53 compared to many other tRNA genes (data not shown), which could partially explain
why their absence has little impact on TGM silencing, which is typically measured
in the context of a plasmid. Alternatively, there could be sufficient redundancy between
Hst1, Sum1, and Sir2 in condensin and cohesin recruitment to tRNA genes.

The diauxic shift, glycolysis and sirtuins

Saccharomyces cerevisiae is a facultative anaerobe that ferments glucose even in the presence of oxygen. During
log phase, genes involved in glycolysis, fermentation and growth-related processes
are highly expressed, whereas genes involved in the tricarboxylic acid cycle and mitochondrial
respiration are repressed [48]. As glucose is depleted, the cells undergo a change in gene expression and metabolism
called the diauxic shift, during which the cells switch from glycolysis to ethanol
catabolism and mitochondrial respiration, and prepare for survival in stationary phase
(G0). Gene upregulation during the diauxic shift is mediated by transcriptional activators
such as Msn2/Msn4 and Gis1, which turn on specific genes in response to the reduction
in nutrients [68]. Specific mechanisms of transcriptional repression during the shift are less well
understood, and are generally believed to be caused by downregulation and inactivation
of the polymerase machinery [68], rather than by specific repressors. The results from our study indicate that Sir2,
Hst1, and Sum1 directly contribute to the repression of genes to which they are bound
across the ORF. The repression does not appear to involve traditional H3 or H4 deacetylation
like that observed at the silenced domains (Figure S5 in Additional file 1), although we cannot rule out other less studied acetylation sites or alternative
lysine modifications being important [69]. One attractive model is that the polymerase machinery or transcription elongation
factors associated with the ORFs during transcription are inactivated by direct deacetylation
during the diauxic shift. While it remains unclear if such factors are acetylated
in yeast, several conserved RNA polymerase and elongation factor subunits have been
identified as acetylated proteins in human cells [70]. In such a model, deacetylation by Sir2 and Hst1 could be partially triggered by
the higher NAD+/NADH ratio that occurs at the onset of the diauxic shift (Figure 5a), with additional nutritional signaling inputs also likely involved. An alternative,
and not mutually exclusive, model is that a repressive higher-ordered chromatin structure
is established at these ORFs during the diauxic shift via the Sir2- and Hst1-mediated
recruitment of condensin and cohesin (see below).

Earlier ChIP-chip studies on Sir2, Sir3, or Sir4 binding sites identified several
euchromatic targets, but either did not test whether such genes were regulated by
the SIR complex, or were unable to detect any expression changes in the SIR mutants
[71-73]. We also did not observe changes in target gene expression during log phase growth
(Figure 4b), but instead uncovered the role in repression of targeted genes downregulated at
the diauxic shift, including glycolytic genes. This regulation of glycolytic genes
by Sir2 and Hst1 is reminiscent of Sirt6 regulation of glycolytic genes in the mouse
[74], where Sirt6 functions as a co-repressor for Hif1α, a critical regulator of nutrient
stress. Sirt6 knock out mice upregulate genes involved in glycolysis and glucose import,
and have reduced mitochondrial respiration activity [74]. During exponential growth in glucose, yeast cells almost exclusively ferment, and
it is only at the diauxic shift when they will normally begin to respire. In this
sense, yeast cells are similar to tumor cells that obtain much of their energy from
aerobic glycolysis (the Warburg effect). Like mouse Sirt6, yeast Sir2 and Hst1 appear
to favor the normal shift to respiration by repressing glycolytic genes when needed.
A key difference is that Sirt6 appears to deacetylate histone tails at promoters [74] to repress genes, while Sir2 and Hst1 associate with the ORFs and likely deacetylate
non-histone proteins. Since the original submission of this manuscript, Sirt6 has
also been found to localize on specific ORFs [75], making the parallel between yeast and mammals even more compelling.

Biochemically, Sirt6 is a histone H3-K9 deacetylase that is required for proper telomere
function and maintenance [76]. More recently, male Sirt6-transgenic mice were shown to have significantly extended
lifespans [13], which does not happen with other sirtuin transgenics, and is consistent with the
effect of increased Sir2 expression in yeast replicative life span [11]. From a life span standpoint, Sir2 appears to be functionally more similar to Sirt6
than Sirt1, a position that is now further supported by our findings that Sir2 and
Hst1 both regulate glycolytic gene expression and are required for proper telomere
maintenance. It is therefore tempting to speculate that these non-traditional functions
for Sir2 could be related to its role in promoting longevity.

Sirtuins and condensin/cohesin

Sir2-dependent recruitment of cohesin to the HM loci and rDNA is well established [40,55,77]. Mutations in cohesin subunits also result in rDNA silencing and recombination suppression
defects [55], as well as impair the silencing boundary function of tRNA genes [78,79]. Interestingly, the tRNAThr boundary element next to HMR-I was previously shown to be important for establishing cohesion across the HMR locus, but silencing of HMR in the absence of the tRNA gene was not sufficient for establishment [80]. The finding that Sir2 contributes to efficient recruitment of cohesin to tRNA genes
helps explain this observation. Sum1 and Hst1 are required for efficient cohesin recruitment
at their shared target locations such as tRNA genes (Figure 7c), and therefore likely contribute to sister chromatid cohesion at those locations.
We also found that high NAD+ levels are critical for cohesin deposition, suggesting that sirtuin activity is involved.
However, a catalytically inactive Sir2 protein can establish cohesion when it is targeted
as a fusion protein adjacent to HMR in place of the tRNAThr gene [56]. Perhaps Hst1 is providing a redundant deacetylation function in this context.

We were compelled to assay for Sir2-mediated recruitment of condensin because of the
highly significant overlap of Sir2, Hst1, and Sum1 binding sites with condensin binding
sites, especially tRNA genes [53,54]. The negative effects of deleting SIR2, HST1, or SUM1 on condensin association with tRNA genes, the rDNA, or specific ORF regions were almost
identical to the effects on cohesin, suggesting that their recruitment to these locations
may involve a common factor. One possibility is the Scc2-Scc4 complex, which loads
the cohesin complex onto chromosomes [81], but has also been suggested to contribute to condensin loading onto tRNA genes [53]. Consistent with this idea, Scc2 enrichment at the rDNA was impaired in an npt1Δ mutant (Figure 8c). A recent Sirt1 study showed that it was involved in the binding of condensin during
mitotic chromosome condensation [82], implying another conserved link between yeast and mammalian sirtuin functions. Taken
together, the requirements for Sir2, Hst1, and elevated NAD+ concentrations in cohesin and condensin deposition at tRNA genes and genes downregulated
at the diauxic shift suggest they may have significant impact on long-range chromosomal
architecture beyond their traditional silenced targets.

Conclusions

The budding yeast S. cerevisiae was used as a model system in identifying functional chromatin binding targets for
all five sirtuins in this organism. A significant amount of overlap was surprisingly
observed between Sir2 and Hst1, which was not shared with Hst2, Hst3, or Hst4. Importantly,
these overlapping binding sites were not the previously described functional targets
of Sir2 (silenced domains) or Hst1 (promoters of specific genes), but were instead
novel targets. Binding of Sir2 and Hst1 at each new target class was functional, including
telomere length maintenance at telomeric repeat clusters, diauxic shift-specific repression
of glycolytic and translation factor genes through the binding to ORFs, and promoting
condensin and cohesin deposition at overlapping positions. Sirtuins have numerous
non-histone targets, but the results from this study highlight the idea that the chromatin
targets of this enzyme class are equally broad and more highly conserved between yeast
and human than previously thought.

Materials and methods

Yeast strains and media

Yeast strains were grown in YPD medium containing 2% glucose as the carbon source.
All yeast growth was performed at 30°C. The strains used in this study were derived
from the GRF167/JB740 strain background used for Ty1 and rDNA silencing studies [37], with the exception being a rap1-17 strain set in the W303 background [44]. All strains are listed in Table S6 in Additional file 1. Deletion strains were created by deleting each ORF and replacing them with either
kanMX4 or natMX4, using one-step PCR-mediated gene replacement, and then PCR confirmation. Myc tagged
strains were produced by fusing 13 copies of the Myc epitope (EQKLISEEDL) at the end
of each gene.

ChIP-sequencing

Log-phase cultures (200 ml) in YPD were cross-linked with 1% formaldehyde for 20 minutes
at 30°C. Cells were pelleted by centrifugation and washed two times with cold Tris-buffered
saline. The cells, in 0.6 ml of FA-140 lysis buffer (50 mM HEPES, 140 mM NaCl, 1%
Triton X-100, 1 mM EDTA, 0.1% SDS, 0.1 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine,
1× protease inhibitor cocktail (Sigma; St. Louis, MO, USA) were lysed with glass beads
in a Mini-BeadBeater (Biospec Products; Bartlesville, OK, USA). The cell lysate was
drawn off the beads, sonicated for 60 cycles (30 s 'on' at high level and 30 s 'off'
per cycle) in a Bioruptor (Diagenode; Denville, NJ, USA) and spun for 10 minutes at
16,000x g in a microcentrifuge. Equivalent amounts of lysate (2.5 mg protein) were
incubated overnight at 4°C with 5 µg of anti-Myc antibody (9E10) and 20 µl of protein
G magnetic beads (Millipore; Billerica, MA, USA). The immunoprecipitated chromatin
was then recovered and the DNA purified using a Magna ChIP™ G Chromatin Immunoprecipitation
Kit (Millipore). The ChIP-sequencing libraries were made using an Illumina ChIP-Seq
DNA Sample Prep Kit (catalogue number IP-102-1001), starting with 0.5 µg of DNA isolated
from the immunoprecipitation step. The libraries were sequenced using an Illumina
GAII system at the University of Virginia Biomolecular Research Facility. Integrative
Genomics Viewer (IGV) was used to visualize the data. The scales were normalized to
the Sir2 read count for each panel, based on the total number of mapped reads recovered.

Standard ChIP assays

Standard ChIP assays to confirm the sequencing results were performed as previously
described [26]. The preparation of chromatin solution from log-phase or post log-phase yeast culture
is the same as described for the ChIP-seq procedure, except anti-myc antibody (9E10)
and each specific antibody was combined with protein A/G conjugated sepharose beads
blocked with salmon sperm DNA in the IP procedure. After incubation of chromatin solution
with antibody and beads at 4°C overnight, the beads containing the immune-complex
were washed (wash 1, twice with 1 ml of FA-140; wash 2, twice with 1 ml of FA-500
(the buffer was the same as FA-140 except that the NaCl concentration was increased
to 500 mM); and wash 3, twice with 1 ml LiCl solution containing 10 mM Tris-HCl, pH
8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium dodecyl sulfate, 1 mM EDTA). DNA was then
eluted from the beads 2 times with 75 µl of elution buffer (5× TE plus 1% SDS). The
combined DNA solution was incubated at 65°C overnight to reverse the cross-linking.
The purified DNA samples were analyzed by quantitative real-time PCR, and the results
normalized with the input DNA PCR signal, and indicated by relative IP in the graphs.

Analysis of ChIP-sequencing reads

Sequence reads for Sir2, Sum1, Hst1, Hst2 libraries (ChIP DNA), and Input DNA were
uniquely mapped to the SGD genome assembly (sacCer2) using BWA version 0.5.7 [83]. Reads that mapped uniquely were filtered on a phred quality score of 20, and were
quantified as Sir2 (20760165), Hst1 (15530594), Sum1 (17613707), Hst2 (2637949), Hst3
(19078082), Hst4 (7210751), and Input (34090115). Repeats were allowed only on the
rDNA locus on chromosome XII. Individual browsable enriched read wig files for Sir2,
Sum1, Hst1, Hst2, and Input DNA were generated from the mapped read files by summing
the number of overlapping reads for every genomic coordinate across the yeast genome.

Significant peaks were determined, relative to the Input DNA, using the BioConductor
package, BayesPeak version 1.2.3 [43]. Subsequently, 1,391, 1,210, 1,527, and 146 peaks were highlighted as significantly
enriched regions for Sir2, Sum1, Hst1, and Hst2 libraries respectively, after the
posterior probability for each peak was required to exceed 0.5. Additional computational
and statistical analyses are described in the Supplemental Information section in
Additional file 1. The ChIP-seq datasets from this study have been deposited in NCBI's Gene Expression
Omnibus, and are accessible through the GEO series accession number [GSE41415].

Southern blotting

Genomic DNA (10 µg) was digested with XhoI at 37°C, and then separated on a 0.7% agarose gel, followed by transfer to an Immobilon-Ny+ membrane (Millipore). The membrane was prehybridized in QuickHyb solution (Stratagene;
Jolla, LA, USA) at 68°C for 20 minutes. To generate the telomeric repeat probe, a
350 bp EcoRI fragment was isolated from pYPLV [84], and labeled with [α32P] dCTP (3,000 Ci/mmol; Perkin Elmer; Boston, Massachusetts, USA) by random priming.
The labeled probe was mixed with 100 µl of sonicated salmon sperm DNA (10 mg/ml),
boiled to denature, and then hybridized to the membrane for 1 hour at 68°C in QuickHyb
solution. The membrane was washed two times at room temperature in 2×-SSC + 0.1% SDS
and once at 60°C in 0.1×-SSC + 0.1% SDS. The washed membrane was exposed to X-ray
film for autoradiography.

Quantitative reverse transcriptase (RT) PCR assays

Synthesis of cDNA from total RNA and PCR reactions with SYBR green PCR master mix
was performed as previously described [26]. The oligonucleotide primer sequences are provided in Table S7 in Additional file
1. The test mRNA transcript levels were normalized to either ACT1 or ALD2. As indicated in some figures, to determine the fold induction, gene transcript levels
in the mutant strains were also normalized to the levels in the wild-type strain.
Results reflect the average fold induction (relative to the induction in the wild-type
strain) from three biological replicates. Where indicated in the figures, the standard
deviation was calculated.

Glucose concentration measurements

The amount of glucose in the growth medium was assayed with a glucose assay kit (Sigma)
using the glucose oxidase system. One milliliter of culture was removed at each time
point from the indicated cultures and centrifuged at 10,000x g for 5 minutes to clarify
the supernatant. The supernatant was further diluted 250-fold with deionized water
and 250 µl of the diluted sample was subjected to the assay. Briefly, the reaction
was started by adding 500 µl of assay reagent that contains o-dianisidine and glucose
oxidase/peroxidase. After reacting for exactly 30 minutes at 37°C, the reaction was
stopped by adding 500 µl of 12 N H2SO4, followed by measuring the absorbance at 540 nM.

Intracellular NAD+ and NADH measurements

To determine the NAD+/NADH ratio, we utilized a fluorescent NAD/NADH detection kit (Cell Technology, Inc;
Mountain View, CA). Yeast cells were inoculated into 100 ml YPD medium and grown at
30°C in a shaker. A quantity of 2 × 106 cells was collected for each time point and then washed twice with 2 ml of phosphate-buffered
saline. After removal of the final supernatant, the cell pellet was resuspended in
200 µl of the NAD or NADH extraction buffer supplied in the kit, and the rest of the
protocol performed according to the manufacturer's instructions.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

ML contributed to conceptualization of the experiments, carried out most of the molecular
biological experiments, and drafting of the manuscript. VV performed bioinformatic
and statistical analysis with the ChIP-seq data and contributed to the writing. KP
contributed to statistical analysis of the ChIP-seq data and diauxic shift gene expression
analysis. SB contributed to conceptualization of the experiments, drafting the manuscript,
and data analysis. JS conceived of the study, contributed to the experimental design,
data analysis, and drafting of the manuscript. All authors read and approved the final
manuscript.

Acknowledgements

We would like to thank Marty Mayo, Natalya Baranova, Neerja Karnani, and Cathleen
Brdlik for advice with ChIP-sequencing, and Patrick Grant for use of their Bioruptor
apparatus. We also thank David Shore and Danesh Moazed for providing strains and plasmids.
Lastly, we thank members of the Smith, Bekiranov, and Grant labs for helpful discussions
and suggestions, as well as Mitch Smith and Marty Mayo for suggestions on the project
and critical comments on the manuscript.